Chakraborty S et al. - In a study to investigate the role of the tumor suppressor genes, TSC1 and TSC2, and other members of this pathway in tumorigenesis of oral squamous cell carcinoma (OSCC), it was found that TSC genes and other members of the mTOR signaling pathway are involved in the pathogenesis of OSCC. LOH and promoter methylation are 2 important mechanisms for downregulation of TSC genes Methods
Expression of genes at the RNA and protein levels was examined by semi-quantitative RT-PCR and western blot analyses, respectively
Loss of heterozygosity (LOH) was studied using matched blood and tumor DNA samples and microsatellite markers from the TSC1, TSC2 and PTEN candidate regions
Methylation status of the TSC2 promoter in tissue samples was examined by combined bisulfite restriction analysis (COBRA)
The effect of promoter methylation on TSC gene expression was studied by treating cells with methyltransferase inhibitor 5-azacytidine
Results
Semi-quantitative RT-PCR analysis showed downregulation of TSC1, TSC2, EIF4EBP1 and PTEN, and upregulation of PIK3C2A, AKT1, PDPK1, RHEB, FRAP1, RPS6KB1, EIF4E and RPS6 in tumors
A similar observation was made for AKT1 and RPS6KB1 expression in tumors at the protein level
Investigation of the mechanism of downregulation of TSC genes identified LOH in 36.96% and 39.13% of the tumors at the TSC1 and TSC2 loci, respectively
No mutation was found in TSC genes
A low LOH rate of 13% was observed at the PTEN locus
Treatment of an OSCC cell line with the methyltransferase inhibitor 5-azacytidine showed a significant increase in expression of TSC genes, suggesting methylation of their promoters
The 5-azacytidine treatment of non-OSCC HeLa cells showed a significant increase in expression of the TSC2 gene only
In order to confirm the results in pt tumor samples, the methylation status of the TSC2 gene promoter was examined by COBRA
Results suggested promoter hypermethylation as an important mechanism for its downregulation
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